Abstract W MP49: Classifying M1/M2 Monocytes by JAK/STAT Activation After Murine Intracerebral Hemorrhage
Background: The immune response following intracerebral hemorrhage (ICH) is an important component of secondary injury that requires further study. Flow cytometry is used to quantify and phenotype recruited leukocytes after murine ICH. Current methods using density gradients have long processing times that reduce the ability to detect phosphorylation states and often yield few neutrophils. Here, we demonstrate the utility of a new method for homogenizing brains and staining for intracellular signaling proteins. This technique results in improved cell recovery and allows us to more precisely classify monocyte polarization states as either M1 pro-inflammatory or M2 anti-inflammatory. Methods: Mouse brains were digested to single-cell suspensions 1-10 days after collagenase ICH. To compare methods, brains were centrifuged on a percoll density gradient or simply homogenized on ice with phosphatase inhibitors. The latter samples were stained extracellularly, fixed with paraformaldehyde, permeabilized in methanol, and then stained intracellularly for phosphorylated STAT molecules (pSTATs). Results: This new method resulted in better neutrophil recovery, yielding 12,190 ± 5,603 neutrophils vs. 1,576 ± 458 neutrophils with percoll at day 3 (p<0.02; n=3-5). No differences were seen in the numbers of monocytes, T cells, or microglia collected between the two methods (p>0.05; n=3-5). Intracellular staining for pSTATs allowed us to identify activated components of the JAK/STAT signaling pathway. All monocytes had high pSTAT1 at day 1, but it steadily declined to day 10. Ly6Clow and Ly6C– monocytes increased pSTAT6 over time, whereas Ly6Chi monocytes did not. Conclusions: This study demonstrates an improved method for recovering leukocytes from mouse brains and staining for pSTATs. Using this technique, we show that Ly6Chi monocytes, which are known to worsen ICH disability, lack pSTAT6 and display an early pro-inflammatory M1 phenotype. In contrast, the Ly6Clow and Ly6C– monocytes both elevate pSTAT6 over time, suggesting they become M2 polarized and may contribute to later repair. Decreasing M1 monocyte polarization and accelerating M2 polarization could be accomplished by targeting pSTATs and may represent a novel treatment strategy in ICH.
Author Disclosures: M.D. Hammond: None. E.C. Lorenzo: None. R.A. Taylor: None. Y. Ai: None. L.H. Sansing: None.
This research has received full or partial funding support from the American Heart Association, Founders - Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont.
- © 2015 by American Heart Association, Inc.